Growth and Aspartate Kinase Activity in Wheat Cell Suspension Culture: Effects of Lysine Analogs and Aspartate-Derived Amino Acids

The effects of lysine analogs and aspartate-derived amino acidson the growth of wheat cell suspension culture were studied.S-(2-Aminoethyl)-L-cysteine (AEC), -hydroxylysine (DHL) andtrans-lysene caused complete growth inhibition at 1.0 mM. Thegrowth inhibition of lysine analogs were, in the order of decreasingeffectiveness; AECDHL, trans-lysene>oxalysine, homolysineand lysyne. cis-Lysene and methyllysine were not inhibitoryeven at concentrations of 10 mM. Lysine effectively relievedgrowth inhibition induced by the lysine analogs. Lysine plusthreonine showed concerted inhibition, which was relieved bythe addition of methionine. Activity of aspartate kinase extracted from wheat cell suspensionculture was strongly inhibited by L-lysine; 0.75 to 1 mM oflysine was required for half-maximal inhibition. Threonine andmethionine, individually or in combination with lysine, showedno inhibitory effect on the enzyme activity. S-Adenosylmethionine,when added with lysine in equimolar concentrations, enhancedthe feedback inhibition by lysine, lowering the concentrationof lysine for half-maximal inhibition to 0.13 mM. The aspartatekinase isolated from the cells cultured in the presence of 5mM lysine did not differ in regulatory properties from the enzymefrom the cells cultured without lysine. AEC at 5 mM inhibitedthe enzyme activity by 50%. Other lysine analogs were not inhibitoryto the enzyme activity even at 10 mM. Growth inhibition of wheat suspension culture by aspartate-derivedamino acids and lysine analogs were discussed in relation totheir inhibitory effects on aspartate kinase activity. (Received October 25, 1985; Accepted February 26, 1986)     

Cloning and expression of human lymphotoxin mRNA derived from a human T cell hybridoma

We cloned human lymphotoxin (LT) cDNA from a cDNA library prepared from phorbol myristate acetate (PMA) and Con A-stimulated human T cell hybridoma (AC5-8) cells, The nucleotide sequence of the cDNA insert in the plasmid, pLT13, was determined and compared with those of peripheral blood mononuclear cell derived LT cDNA and the LT gene. Four stretches, containing 14 nucleotides in total, were different among the three sequences. The differences included one base change from C to A in the coding region. Because this change was expected to result in a change in the amino acid at position 26 from Thr to Asn, we constructed an E. coli expression plasmid (pLT13tac6-8.2) and a mammalian cell expression plasmid (pSV2-LT) in order to confirm that pLT13 contains the coding sequence of human LT. Both plasmids were found to synthesize active LT molecules after transfection into JM105 and COS-1 cells, respectively, and their lytic activities were found to be completely neutralized by anti human LT serum. Using an insertion mutant and a deletion mutant, we examined the role of the C terminal domain in the LT activity. The results obtained strongly suggested that the intactness of the C terminal less than 10 residues is required for the LT activity.     

Biochemical Characterization of α-Adrenergic Receptors in Human Brain and Changes in Alzheimer-Type Dementia

Abstract Using ligand binding techniques, we studied α-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the α-adrenergic antagonists [³H]prazosin and [³H]yohimbine to membranes of human brains exhibited characteristics compatible with α₁- and α₂-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [³H]prazosin and 5.5 nM for [³H]yohimbine. [³H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [³H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [³H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [³H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that α₁ and α₂-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD.     

(6R)-Tetrahydrobiopterin Increases the Activity of Tryptophan Hydroxylase in Rat Raphe Slices

The effects of (6R)- and (6S)-tetrahydrobiopterin (BPH4), tetrahydroneopterin, and 6-methyltetrahydropterin on the activity of tryptophan hydroxylase were investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measurement of 5-hydroxytryptophan (5-HTP) formation under inhibition of aromatic L-amino acid decarboxylase with use of HPLC-fluorometric detection. (6R)-BPH4 (the naturally occurring form) at 42 microM, tetrahydroneopterin at 50 microM, and 6-methyltetrahydropterin at 100 microM increased tryptophan hydroxylase activity to 350, 145, and 146% of control values, respectively. (6S)-BPH4, however, had no significant effects on tryptophan hydroxylase activity. These results suggest that tryptophan hydroxylase is subsaturating in vivo for the naturally occurring cofactor, (6R)-BPH4, and that the concentration of (6R)-BPH4 may play an important role for the regulation of tryptophan hydroxylase activity in vivo.     

An oxygen-evolving complex with a simple subunit structure - ‘a water-plastoquinone oxidoreductase” - from the thermophilic cyanobacterium Synechococcus sp.

An oxygen-evolving complex has been highly purified from the thermophilic cyanobacterium Synechococcus sp. The complex, which reproducibly showed 5 major polypeptide bands of 47, 40, 35, 30 and 9 kDa on SDS-polyacrylamide gel electrophoresis and contained 3.2 Mn per Q_A, had an oxygen-evolving activity of 300–400 μmol/mg chl per h in the presence of 5 mM MnCl₂; or CaCl₂. The complex most likely represents a minimum functional unit of the photosynthetic oxygen evolution.     

Purification and characterization of thylakoid-bound Mn-superoxide dismutase in spinach chloroplasts

Thylakoid-bound superoxide dismutase (SOD; EC 1.15.1.1) was solubilized by Triton X-100 from spinach and purified to a homogeneous state. The molecular weight of thylakoid-bound SOD was 52000; the enzyme was composed of two equal subunits. Its activity was not sensitive to cyanide and hydrogen peroxide, and the isolated SOD contained Mn, but neither Fe nor Cu. Thus, the thylakoid-bound SOD is a Mn-containing enzyme. The subunit molecular weight of thylakoid Mn-SOD is the highest among Mn-SODs isolated so far, a fact which might reflect its binding to the membranes.     

The differentiation of HL-60 cells in the synthetic medium induced by GM3 ganglioside

GM3 ganglioside, added exogenously to a promyelocytic leukemia cell line (HL-60 cells) in serum-free synthetic medium, induced differentiation into macrophage-like cells. Macrophagic morphology and function of differentiation-induced cells were determined by cell growth behavior, May-GriJnwald-Giemsa staining, activities of nonspecific esterase, phagocytosis and nitroblue tetrazolium (NBT) reduction. GM3 ganglioside may play a role in triggering differentiation of HL-60 cells into macrophage-like cells.     

In vitro establishment of human fibroblasts of lysosomal diseases,GM1-gangliosidosis and Sandhoff disease,by transformation with origin-minus SV40 DNA

The permanent human cell lines preserving defects of lysosomal enzymes, G_M1-1019-SV and SA-1077-SV, were established from the respective fibroblasts from patients with G_Ml-gangliosidosis and Sandhoff disease by transfection with replication origin-minus simian virus 40 DNA. These ceils grow rapidly without entering senescence during more than 120 population doublings. The activity of -galactosidase in G_M1-1019-SV and of B-N-acetylhexosaminidase in SA-1077-SV was respectively 40- and 180-fold lower than that of normal fibroblasts.     

Formation of giant protoplasts from protoplasts of Pleurotus cornucopiae by the cell wall lytic enzyme

Summary When purified protoplasts of Pleurotus cornucopiae IFO9614 were incubated with a mixture of cell wall lytic enzymes, they were found to increase their size. Their average diameter increased from 4.3 m to 31 m after 65 h incubation at 24° C. The presence of cellulase ONOZUKARS in the enzyme mixture had a significant effect on the formation of giant protoplasts. Regeneration frequency of giant protoplasts in a medium containing 0.5 M sucrose was 3.5%, approximately six times that of normal protoplasts.     

Effect of DN-1417, a thyrotropin releasing hormone analog, on dopaminergic neurons in rat brain

Acute and chronic effects of γ-butyrolactone-γ-carbonyl-histidyl-prolinamide (DN-1417) were investigated on motor activity, dopamine (DA) metabolites and DA receptors in various brain regions of rats. The motor activity, as measured with Automex recorder, was enhanced after a single injection with DN-1417 (20 mg/kg, IP), and the motor stimulating action persisted during 21 daily injections. Acute DN-1417 elevated both homovanillic acid (HVA) and 3,4-dihydroxyphenylacetic acid (DOPAC) levels in 7 brain regions, prefrontal cortex polar, medial and lateral fields, nucleus accumbens, olfactory tubercles, amygdala and striatum. After chronic treatment for 7 days, the acute effect of DN-1417 on DA metabolites disappeared in all regions except for the striatum in which DN-1417 still increased HVA and DOPAC. The response of striatal DA metabolites was also observed after chronic treatment for 21 days. Chronic DN-1417 produced no significant change in ³H-spiperone binding in the prefrontal cortex, nucleus accumbens, olfactory tubercles and striatum, while striatal ³H-DA binding displaced by 30 nM spiperone was enhanced after chronic treatment. These results indicate that DN-1417 interacts with mesocortical, mesolimbic and nigrostriatal DA systems in the different modes of action. The lack of tolerance to motor hyperactivity, however, raises the question as to whether DN-1417-induced hyperactivity may be mediated by the activation of mesolimbic DA neurons. The involvement of nigrostriatal neurons in DN-1417-induced motor hyperactivity is suggested.